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| Status |
Public on Jul 18, 2017 |
| Title |
Pho ChIP-seq in S2 cells |
| Sample type |
SRA |
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| Source name |
s2-cells-pho
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Schneider's Drosophila Line 2 (SL2) cells
cell line: SL2
passages: 10 to 12
chip antibody/selection: Pho antibody
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| Biomaterial provider |
ATCC [D. Mel. (2), SL2] (ATCC® CRL-1963™)
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| Treatment protocol |
Cells were not treated.
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| Growth protocol |
Drosophila melanogaster Schneider S2 cells were cultured in Sf-900 II SFM medium (Invitrogen) supplemented 100 U/ml Penicillin and 100 mg/ml Streptomycin at 25°C. Cells were passaged at 1:4 ratio every two days to keep logarithmic growth.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
~2x107 cells were fixed for 10 min at room temperature by gently mixing in 10 mL crosslinking solution (1% formaldehyde, 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), crosslinking was quenched by adding 2M glycine to a final concentration of 240 mM. The cells were sequentially collected by centrifugation at 700 x g for 10 min and washed trice with 10 mL ice-cold PBS. These cell pellets were sonicated in parallel to obtain chromatin fragments of ~200 to 700 bp, each in 4 mL sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, Roche protease inhibitor cocktail) with a Sonic Dismembrator Model 500 (Fisher Scientific, 10 x 30 seconds on / 45 seconds off cycles, 50% power settings). After sonication, debris was removed by centrifugation at 4°C for 10 minutes at 13,000 rpm, and equal amount of 6M urea was added to the solution then incubated for 10 minutes at 4°C on a rotating wheel. The soluble chromatin was dialysed using a membrane with a molecular weight cut-off of 3.5 kDa, at 4°C against 2 L dialysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 4% glycerol) overnight. Insoluble debris was removed by quick centrifugation (7,500 rpm, 2 min). 120 μg of extract were incubated in a final volume of 1 mL IP buffer (15 mM Hepes pH 7.9 / 200 mM KCl / 1.5 mM MgCl2 / 0.2 mM EDTA pH 8 / 0.25% NP-40 / 20% glycerol / 0.3 mM DTT / 1x "Complete" protease inhibitor cocktail / 1 mM PMSF) with 1:100 anti-Pho antibody (kindly provided by Judith Kassis1) for 12 hours at 4°C. 100 µL protein A/G agarose beads (Calbiochem), previously blocked with 1 mg/mL BSA, was added to each chromatin at 4°C overnight. The beads were sequentially washed trice with 5-10 volumes of 6 M urea/2 M thiourea/10 mM HEPES (pH 8.0), PBS, and 1% SDS/PBS. Centrifugation of the beads between washing steps was carried out (7,500 rpm, 2 min).
The libraries were prepared according to the manufacturer's instructions using the NEBNext kit (E6200). Briefly, DNA was fragmented by sonication to a maximum of 300 bp. Next the ends of the fragments were repaired with a combination of fill-in reactions and exonuclease activity to produce blunt ends that were then tailed with an A-base. Illumina-specific Adaptors were ligated followed by removal of unligated adaptors using AMpure XP beads (Beckman Coulter). Finally a PCR with 12-15 cycles for both kits was performed to enrich final adaptor-ligated fragments. Quality and quantity were assessed on an Agilent Bioanalyzer Chip DNA 7500 or High Sensitivity. For all libraries, sequencing was performed on the MiSeq platform, using v2, 300-cycle reagent kits (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
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| Data processing |
ChIP-seq reads were aligned to the mm9 genome assembly using bwa-mem or bwa-bwasw algorithm version 0.7.5a-r405.
Bam-format alignment files were generated with Samtools version 0.1.19-96b5f2294a.
Multiple runs were merged with Samtools.
Duplicate read-pairs were removed with Samtools.
BDG files generated with macs version1.4
Genome_build: dm3
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| Submission date |
Jul 18, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
David Vocadlo |
| E-mail(s) |
dvocadlo@sfu.ca
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| Organization name |
Simon Fraser University
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| Department |
Chemistry
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| Street address |
8888 University Drive
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| City |
Burnaby |
| State/province |
British Columbia |
| ZIP/Postal code |
V5A 1S6 |
| Country |
Canada |
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| Platform ID |
GPL16479 |
| Series (1) |
| GSE84502 |
Genome-wide maps of GlcNAc bound proteins and Pho ChIP-seq in Drosophila S2 cells and pupae. |
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| Relations |
| BioSample |
SAMN05413710 |
| SRA |
SRX1958586 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2238595_s2-cells-pho.bedgraph.gz |
57.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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